Abstract
Homologous recombination (HR)-directed DNA double-strand break (DSB) repair enables template-directed DNA repair to maintain genomic stability. RAD51 recombinase (RAD51) is a critical component of HR and facilitates DNA strand exchange in DSB repair. We report here that treating triple-negative breast cancer (TNBC) cells with the fatty acid nitroalkene 10-nitro-octadec-9-enoic acid (OA-NO2) in combination with the antineoplastic DNA-damaging agents doxorubicin, cisplatin, olaparib, and γ-irradiation (IR) enhances the antiproliferative effects of these agents. OA-NO2 inhibited IR-induced RAD51 foci formation and enhanced H2A histone family member X (H2AX) phosphorylation in TNBC cells. Analyses of fluorescent DSB reporter activity with both static-flow cytometry and kinetic live-cell studies enabling temporal resolution of recombination revealed that OA-NO2 inhibits HR and not nonhomologous end joining (NHEJ). OA-NO2 alkylated Cys-319 in RAD51, and this alkylation depended on the Michael acceptor properties of OA-NO2 because nonnitrated and saturated nonelectrophilic analogs of OA-NO2, octadecanoic acid and 10-nitro-octadecanoic acid, did not react with Cys-319. Of note, OA-NO2 alkylation of RAD51 inhibited its binding to ssDNA. RAD51 Cys-319 resides within the SH3-binding site of ABL proto-oncogene 1, nonreceptor tyrosine kinase (ABL1), so we investigated the effect of OA-NO2-mediated Cys-319 alkylation on ABL1 binding and found that OA-NO2 inhibits RAD51-ABL1 complex formation both in vitro and in cell-based immunoprecipitation assays. The inhibition of the RAD51-ABL1 complex also suppressed downstream RAD51 Tyr-315 phosphorylation. In conclusion, RAD51 Cys-319 is a functionally significant site for adduction of soft electrophiles such as OA-NO2 and suggests further investigation of lipid electrophile-based combinational therapies for TNBC.
Highlights
Homologous recombination (HR)-directed DNA doublestrand break (DSB) repair enables template-directed DNA repair to maintain genomic stability
Emerging evidence has revealed that triple-negative breast cancer cell growth, migration, and invasion are suppressed by treatment with the electrophilic nitro-fatty acid derivative nitro-oleic acid (10-nitro-octadec-9-enoic acid (OA-NO2)4) through modulation of NF-B signaling, whereas nontumorigenic breast epithelial cells are resistant to the effects of OA-NO2 because of more intact mechanisms for maintaining redox homeostasis
MDA-MD-231 cells were implanted into the mammary gland of mice, and when tumors reached a volume of 100 mm3, mice were treated with 15 mg/kg nonelectrophilic fatty acid oleic acid (OA) or OA-NO2 by gavage for 4 weeks
Summary
Homologous recombination (HR)-directed DNA doublestrand break (DSB) repair enables template-directed DNA repair to maintain genomic stability. We report here that treating triple-negative breast cancer (TNBC) cells with the fatty acid nitroalkene 10-nitro-octadec-9-enoic acid (OA-NO2) in combination with the antineoplastic DNA-damaging agents doxorubicin, cisplatin, olaparib, and ␥-irradiation (IR) enhances the antiproliferative effects of these agents. A. N.); University of Pittsburgh Department of Pharmacology and Chemical Biology and William C. de Groat predoctoral fellowships OA-NO2 was identified to suppress IR-induced RAD51 foci formation, inhibit RAD51 binding to ssDNA, decrease HR, induce phosphorylation of Ser-139 H2AX (␥H2AX), disrupt RAD51–ABL heterodimerization, and decrease RAD51 Tyr-315 phosphorylation These observations reinforce the concept that reactive species induce genomic perturbations in part via the disruption of HR and reveal a novel therapeutic strategy: that redox-derived soft electrophiles sensitize cancer cells to DNA-directed therapeutic strategies such as IR, cisplatin, and doxorubicin
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